EP1810036A1 - Colorimetric strip containing coomassie blue for semi-quantitation of albumin - Google Patents
Colorimetric strip containing coomassie blue for semi-quantitation of albuminInfo
- Publication number
- EP1810036A1 EP1810036A1 EP05810435A EP05810435A EP1810036A1 EP 1810036 A1 EP1810036 A1 EP 1810036A1 EP 05810435 A EP05810435 A EP 05810435A EP 05810435 A EP05810435 A EP 05810435A EP 1810036 A1 EP1810036 A1 EP 1810036A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- amount
- albumin
- sample
- test strip
- color
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green
Definitions
- This invention relates to an apparatus and methods for detecting albumin in urine, which is predictive of renal disease and/or renal complications of a disease, using a Coomassie Blue based assay to estimate total urinary albumin including immunoreactive and immunounreactive albumin. More particularly, the invention relates to a rapid, semi ⁇ quantitative test strip and methods for estimating total urinary albumin.
- Dye-based assays such as dye-based test strips for protein detection also fail to detect immunounreactive albumin in urine.
- dyes commonly used to detect albumin e.g., sulfonephthalein dye which is used on Bayer's Microalbustix, and Bayer's Clinitek do not react with immunounreactive intact albumin. Consequently, these test strips fail to detect albumin in urine during the early stages of renal disease or malfunction or early stages of cardiovascular disease.
- the invention provides a test strip for detecting and semi-quantitating the amount of albumin in a bodily sample, such as a urine sample.
- the test strip comprises a reagent area having an amount of Coomassie Blue dye immobilized and dried onto it such that when the pad is dipped into the sample or a sample is applied directly to the reagent area, a color change occurs.
- the resulting color is read against a color standard which relates the amount of protein detected by the Bradford assay to the standard amount of intact albumin detected by HPLC, which is an accurate measure of amount of albumin in a test sample.
- the resulting color is directly proportional to the amount of intact albumin present in the sample.
- a colorimetric test strip for detecting and semi-quantitating the amount of total albumin in a bodily sample comprising a test strip matrix, and at least one reagent area disposed on the test strip matrix comprising Coomassie Blue dye, wherein the at least one reagent area changes color shade after exposure to the sample, wherein the amount of total albumin is determined by comparing the at least one reagent area color shade after exposure to the sample to at least one reference color, said at least one reference color correlating the amount of protein determined by Bradford assay to the amount of albumin determined by HPLC.
- Figure 1 shows a perspective view of a test strip of the invention.
- (1) is the test strip matrix; (2) test pad (reagent area).
- Figure 2(A-B) illustrates the variation of the amount of protein determined by the Bradford assay (expressed as a ratio of total protein to creatinine in units of mg/mmol) compared to the ratio of total albumin determined by HPLC (expressed as the ratio of albuminto creatinine) for urine samples containing relative low amounts of total albumin ( Figure 2A) and in urine samples containing relative high amounts of total albumin ( Figure 2B).
- Figure 3 illustrates the average result of color discrimination on a test strip after urine testing and five minutes of color development.
- the present invention provides a simple and accurate colorimetric test strip and method for measuring urinary albumin in a bodily fluid such as urine. Briefly, one or more reagent areas of the test strip of the invention is dipped into a sample, e.g., urine sample or a small amount of sample is applied to the test strip onto the reagent area(s) and color development at the reagent area(s) is compared to a reference color or colors to determine an estimate of the amount of albumin present in the sample.
- the present inventor has discovered that the Bradford assay, which is a colorimetric test tube assay for detecting protein and which contains Coomassie Blue as a protein indicator, detects both immunoreactive and immunounreactive forms of albumin.
- the Bradford assay has been adapted to a test strip which detects urinary albumin at a sensitivity that strongly correlates with the results obtained using an HPLC-based assay, i.e., AccuminTM.
- Coomassie Blue-based Bradford assay In addition to detecting immunoreactive and immunounreactive forms of albumin in urine, the Coomassie Blue-based Bradford assay also detects any other protein that is present. However, given that other proteins are at relatively low concentration compared to albumin in urine, Coomassie Blue is useful for the detection of total urinary albumin using a correction factor developed by the inventor that correlates the amount of protein detected by Coomassie Blue with the amount of urinary albumin detected by an HPLC-based assay. The correction factor is used to provide color standards against which the test strip color development is compared to determine an estimate of the amount of albumin in a urine sample.
- the test strip of the invention is designed to utilize the Bradford Reagent, which produces a colorimetric change when reacted with proteins in a biological solution in conjunction with a correction factor which relates the amount of protein detected by the Bradford assay to the corresponding amount of albumin detected by HPLC.
- the Bradford reagent is preferably dried and stabilized onto a test pad adhered to at least one end of a solid support matrix.
- the support matrix may be composed of any suitable material such as plastic or polystyrene, for example.
- the change in color of the reagent area on the test pad upon reacting with protein is directly proportional to the concentration of protein in the patient sample.
- the color intensity that develops on the test strip may be determined visually or by a reflectance-based reader, for example.
- the color intensity that develops on the test strip is compared to at least one, and preferably at least two standard color shades that correspond to a range of albumin concentration determined by application of a correction factor.
- the test strip may be manufactured in any size and shape, but in general the strip matrix is longer than wide and is preferably made of firm or stiff materials, e.g., polyethylenesulfone (Supor), cellulose, mixed synthetic fibers, polycarbonate, polypropylene material, charged membranes and glass fibers, and the like.
- the test strip matrix may be washed with acid or base to remove undesired material to reduce background or endogenous color.
- the test strip is a plastic or polystyrene backed strip having a reagent test pad adhered to at least one end. An embodiment of a test strip of the invention is shown in Figure 1.
- the test pad onto which the Bradford Reagent is absorbed and dried is preferably made of a membrane material that shows minimal background color.
- the test pad may be constructed of acid or base washed materials in order to minimize background color. Background color has been observed with several types of membrane materials tested under various conditions (such as pH, concentration etc)- including polyethylenesulfone, cellulose,mixed synthetic fibers, polycarbonate, polypropylene and glass fiber materials.
- a reflectance meter having an LED in the range of about 590 to about 660 nm, rather than visually.
- a glass fiber test membrane is used to form the test strip matrix. Because glass membrane tends to absorb material non-uniformly, the addition of polymers, or gels such as polyethylene glycol, polyvinylpyrolidine (PVP), Klucel, Luviskol K-30, or Bioterg A-40 in the reagent area is preferred in order to coat the test pad membrane uniformly and produce uniform color, which is preferred for good color discrimination in performing tests.
- polymers, or gels such as polyethylene glycol, polyvinylpyrolidine (PVP), Klucel, Luviskol K-30, or Bioterg A-40 in the reagent area is preferred in order to coat the test pad membrane uniformly and produce uniform color, which is preferred for good color discrimination in performing tests.
- the active color-changing protein indicator of the Bradford Reagent is the dye, Coomassie Brilliant Blue G-250 ("Coomassie Blue”)- Coomassie Blue in the appropriate acid medium provides a protein assay reagent having a sensitivity approximately 100 times greater than other protein detection methods, including, the biuret (Mokrasch, L.C., and McGilvery, r. W. (1956) J. Biol. Chem. 221, 909-917) and conventional dye binding techniques, and about three to five times that of the Lowry method (Lowry, Oh. H., Rosenbrough, NJ., Farr, A.L., and Randall RJ. (1951) J. Biol. Chem. 193, 265-275). (U.S. Patent No.
- the acid ingredient of the Bradford Reagent preferably has a pKa of from 0 to 4, more preferably from 1 to 2, and the resultant dye-containing solution preferably has a pH of from -1 to 1, preferably -0.5 to 0.5.
- Suitable acids include phosphoric acid and other acids with a pKa from 1-2 which do not result in protein precipitation.
- Typical candidates include acetic, periodic, phosphoric, selenic, sulfurous, maleic, oxalic, dichloroacetic acids and the like, and any combination of one or more.
- Phosphoric acid is especially preferred.
- phosphoric acid, acetic acid or maleic acid are used.
- the Coomassie Blue and acid solution may be dissolved in any aqueous medium that preferably does not contain surfactants, detergents, or exceedingly strong alkali, preferably water.
- the final concentration of the Coomassie Blue dye in the Bradford Reagent is preferably from about 0.001 to about 0.1% (w/v), more preferably from about 0.005 to about 0.05% (w/v); while that of the acid is preferably from about 4 to about 12% (w/v), more preferably from about 7.5 to about 9.5% (w/v).
- the order of addition of the dye and acid is immaterial and both may be added directly to the aqueous medium or may be added to separate portions of the medium and thereafter mixed.
- the Bradford Reagent which is dried onto the test strip of the invention further comprises a buffer to prevent color changes resulting from changes to pH in the absence of urinary protein.
- Coomassie Blue is a pH indicator and contains an ionizable group which is displaced in the presence of protein to provide a detectable color change. This is the same color change that Coomassie Blue would undergo under the influence of a pH change.
- a buffer such as for example, maleic acid, phosphoric acid, and the like, in the Bradford Reagent to thereby avoid a pH increase which might cause a color change in the absence of protein, thereby resulting in a false positive result.
- the Bradford Reagent which is dried onto the test strip further comprises wetting agents to reduce brittleness of the test pad membrane.
- wetting agents include TritonX-100, Bioterg, glycerol, 0 Tween, and the like.
- the concentration of the Bradford Reagent required on a dry pad is sufficient to allow discrimination in color development between 10 to 200mg/L albumin concentration.
- the test strip contains about 0.01 to about 1% of the Bradford Reagent, with a preferred range of about 0.01% to about 0.03%.
- the Bradford Reagent can be applied to the test strip by any method known in the art.
- membranes from which the test strip pad are made may be dipped into a solution of the Bradford Reagent and dried, preferably in an oven at about 45 to about 75 0 C for about five to about 45 minutes.
- reagents are dried onto the membrane at about 60°C within about 30 to about45 minutes.
- the membranes onto which the Bradford Reagent has been applied can be cut in to any dimension to be affixed to a test strip holding device.
- a test pad having dimensions of about 5mm x 5mm or the like is fixed onto a 5mm x 40mm in plastic or polystyrene matrix which forms the test strip.
- two test pads may be adhered on a single test strip to allow discrimination between high and low protein levels, as a procedural control, to achieve appropriate controlled acidic environments or to test other analytes such as creatinine or glucose in the test sample.
- the amount of total albumin in a sample is determined by comparing the resulting color on the test pad area(s) of a test strip after dipping the test pad into a sample or applying the sample to the test pad(s) area of the test strip to at least one color standard.
- the color standard of the invention was determined by measuring the amount of total protein in urine samples containing a wide range of total albumin using the Bradford assay and measuring the amount of albumin in the urine samples by HPLC.
- the amount of protein detected by the Bradford assay and HPLC is preferably normalized by expressing the amounts of protein or albumin as a protein(albumin) to creatinine ratio.
- Quantitaive albumin excretion is preferably expressed as a ratio of albumin to creatinine to allow for variation in urine flow rates which in turn can alter albumin concentration.
- the amount of total protein measured by the Bradford Assay was plotted against the amount of albumin measured by HPLC for test samples containing relative high and low amounts of total albumin (imrnunoreactive and immunounreactive albumin).
- the results of the comparison of Bradford protein and HPLC total albumin are shown in 2 (A and B).
- the drawn lines in the figures represent the confines of the data.
- Figures 2A and B illustrate the variation of the amount of protein as determined by the Bradford assay (expressed as a ratio of Bradford to creatinine with units of mg/mmol) as compared to the ratio of total intact albumin as determined by HPLC (expressed as the ratio HPLC to creatinine) for urine samples from different individuals containing relative low amounts of total intact albumin and urine samples containing relative high amounts of total intact albumin.
- Normal albumin excretion has a value of albumin/creatinine ratio ⁇ 3.5.
- Quantitative albumin excretion is preferably expressed as a ratio to creatinine to allow for variation in urine flow rate which in turn will alter albumin concentration.
- the range of the conversion factor may preclude accurate quantitative estimation of total urinary albumin by the Bradford assay, but this conversion range is useful for semi- quantitaive estimation of total urinary albumin in the test strip format.
- the results show that normal albumin excretion observed in healthy individuals has an albumin: creatinine ratio value of less than 3.5 by HPLC (see Figure 2), which corresponds to a Bradford protein: creatinine ratio of less than 5.5.
- the data show that abnormal excessive albumin excretion results in an albumhrcreatinine ratio of greater than 3.5 by HPLC measurement, which corresponds to a Bradford proteinxreatinine ratio of greater than 5.5.
- the conversion factor is used to develop one or more reference colors for comparison with the color development observed on the test strip after exposure to a test sample.
- the color shade of the reference colors correspond to the predetermined numerical value for albumin.
- albumin content in a sample is determined to be in the healthy range, i.e., less than 3.5 mg/mM (HPLC) or 5.5 mg/mM Bradford) by comparing the color shade that develops on the test pad with a range of reference colors provided with the strip.
- albumin content in a sample is determined to be in the abnormal, i.e., greater than 3.5 mg/mM (HPLC) or greater than 5.5 mg/mM Bradford) by comparing the color shade that develops on the test pad with a range of reference colors provided with the strip.
- the reference color shade corresponding to a particular value for albumin content is the darkest shade of color development detected for that particular value.
- the conversion factor of Bradford to total intact albumin may be used to design a semi-quantitative estimate of total intact albumin in other formats, such as lateral flow devices or similar devices that are made to come in contact with urine. Such devices may have the Bradford reagent in a format which undergoes a color change depending on the amount of protein in the urine. This color change can be converted to total intact albumin estimate based on prior calibration of the Bradford assay with total intact albumin as determined by HPLC.
- a range of conversion factors to estimate total albumin content of a urine sample may be developed for use with other assays based on protein detecting dyes such as pyrogallol, biuret, bicinconinic and sulfosalicylic acid, for example.
- the amount of total protein in a sample is measured using the dye-based assay, which may be in a dipstick format, and the amount of total protein detected is compared to the amount of albumin detected by HPLC.
- the assays are carried out as above, using different test samples containing high and low concentrations of albumin and the results of the assays are compared.
- a range of conversion factors is developed based on the results.
- test strips of the present invention include the following features.
- test strips were prepared:
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- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
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- Immunology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
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Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/967,276 US20060084175A1 (en) | 2004-10-19 | 2004-10-19 | Colorimetric strip containing coomassie blue for semi-quantitation of albumin |
PCT/US2005/037266 WO2006044811A1 (en) | 2004-10-19 | 2005-10-18 | Colorimetric strip containing coomassie blue for semi-quantitation of albumin |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1810036A1 true EP1810036A1 (en) | 2007-07-25 |
Family
ID=35744658
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP05810435A Withdrawn EP1810036A1 (en) | 2004-10-19 | 2005-10-18 | Colorimetric strip containing coomassie blue for semi-quantitation of albumin |
Country Status (4)
Country | Link |
---|---|
US (1) | US20060084175A1 (en) |
EP (1) | EP1810036A1 (en) |
CA (1) | CA2585816A1 (en) |
WO (1) | WO2006044811A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016038505A2 (en) | 2014-09-08 | 2016-03-17 | Indian Institute Of Science | Electrochemical biosensor and a method of sensing albumin and its complexes |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2687846B1 (en) * | 2008-06-13 | 2016-10-12 | Alt Bioscience, Llc. | Device for rapid determination of disease-associated thiol compounds |
JP5351585B2 (en) * | 2009-03-31 | 2013-11-27 | シスメックス株式会社 | Kidney disease diagnosis support device and computer program |
CN102893159A (en) * | 2010-05-17 | 2013-01-23 | 宝洁公司 | Methods of detecting and demonstrating hair damage via detection of protein loss |
US10132803B2 (en) | 2015-08-07 | 2018-11-20 | Xerox Corporation | Sulfonated polyester-metal nanoparticle composite toner for colorimetric sensing applications |
JP7327985B2 (en) * | 2019-04-25 | 2023-08-16 | 株式会社明治 | Protein detection method |
CN114324210A (en) * | 2021-12-30 | 2022-04-12 | 上海瑞邦生物材料有限公司 | Method for detecting protein load of porous calcium phosphate |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4023933A (en) * | 1976-06-10 | 1977-05-17 | The University Of Georgia | Protein-assay reagent and method |
US4219337A (en) * | 1978-04-27 | 1980-08-26 | The Medical College Of Wisconsin | Assay for proteins and polypeptides |
US5096833A (en) * | 1990-05-29 | 1992-03-17 | Miles Inc. | Method and device for determining protein using carrier matrix composed of urethane, water insouble inorganic compound and insoluble organic compound and method of making the device |
DE69213826T2 (en) * | 1991-06-06 | 1997-01-30 | Bayer Ag | Test strips with merocyanine and nitro or nitroso substituted polyhalogenated phenolsulfonphthaleins as protein indicators |
CA2451611A1 (en) * | 2001-06-25 | 2003-01-03 | Bayer Healthcare Llc | Total protein detection methods and devices at low ph |
-
2004
- 2004-10-19 US US10/967,276 patent/US20060084175A1/en not_active Abandoned
-
2005
- 2005-10-18 CA CA002585816A patent/CA2585816A1/en not_active Abandoned
- 2005-10-18 EP EP05810435A patent/EP1810036A1/en not_active Withdrawn
- 2005-10-18 WO PCT/US2005/037266 patent/WO2006044811A1/en active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2006044811A1 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016038505A2 (en) | 2014-09-08 | 2016-03-17 | Indian Institute Of Science | Electrochemical biosensor and a method of sensing albumin and its complexes |
Also Published As
Publication number | Publication date |
---|---|
CA2585816A1 (en) | 2006-04-27 |
US20060084175A1 (en) | 2006-04-20 |
WO2006044811A1 (en) | 2006-04-27 |
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